Human neutrophils were isolated from human whole blood by using 1-Step Polymorph (Accurate Chemical & Scientific Corp., Westbury, NY) according to the manufacturer’s instructions. Human monocytes were isolated by RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies, London, UK). Human PBMC were prepared by the Ficoll-Hypaque centrifugation method.

For the calcium flux assay, both human neutrophils and monocytes were treated with formate, acetate and propionate (Sigma, St. Louis, MO, USA) for 5-10 min. For measurement of PGE₂, cytokines and chemokines, human monocytes and PBMC cells were incubated with formate, acetate, propionate and butyrate (Sigma, St. Louis, MO, USA) overnight.

TaqMan primers and probes were designed with Primer Express software and purchased from ABI (Applied Biosystems, Foster City, CA, USA). The sequences of the human primers and probes for GPR41 and GPR43 are shown in Table 1. Quantitative real-time PCR was carried out with an ABI Prism 7900HT Sequence Detection System. The PCR reactions were prepared using the components from the iScript Custom One-Step RT-PCR Kit with ROX and assembled according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). The final concentrations of the primers and probe in the PCR reactions were 200 nmol/L and 100 nmol/L, respectively. The fluorogenic probes were labeled with 6-carboxyfluorescein as the reporter and 6-carboxy-4,7,2,7’-tetramethylrhodamine as a quencher. Each 10 μL PCR reaction contained 10 ng of total RNA. The RT-PCR reactions were performed in triplicate in a 384-well plate according to the following protocol: one cycle for 10 min at 50°C, followed by one 5 min cycle at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. A eukaryotic 18S rRNA endogenous control probe/primer set (ABI) was used as an internal control for RNA quality.

The PCR data were quantified, based on a 12-point standard curve generated using 4-fold serial dilutions of a cDNA containing the gene of interest. The 4-fold dilutions began at 20 000 fg. This procedure provides an absolute quantification of the amounts of GPR41 and GPR43 mRNA in a given sample.

Intracellular Ca²⁺ assays were carried out as previously described with modifications[26]. Freshly isolated human neutrophils were resuspended at 1 × 10⁶ cells/mL in cell culture medium (RPMI 1640 with 2 mmol/L GlutaMAX and 5% fetal bovine serum) and incubated at 37°C, 50 mL/L CO₂, for 1 h. Cells were spun and resuspended at 1 × 10⁶ cells/mL in a 1:1 mixture of cell culture medium and Calcium-4 no wash dye (Molecular Devices, Sunnyvale, CA, USA) containing a final concentration of 5 mmol/L probenecid (Sigma). The cells were seeded (50 000/well) into poly-D-lysine-coated 384-well, black-wall, clear bottom microtiter plates (Becton Dickinson). Cells were sedimented in plates by spinning down briefly at low speed with no brake then incubated at 37°C, 50 mL/L CO_2, for 1 h before assaying on a FLIPR-384 (Molecular Devices). Freshly isolated human monocytes were resuspended at 1 × 10⁹ cells/L in a 1:1 mixture of cell culture medium and Calcium-3 no wash dye containing a final concentration of 2.5 mmol/L probenecid. The cells were seeded (50 000/well) into poly-D-lysine coated 384-well plates. Cells were dye loaded for 45 min at 37°C, 50 mL/L CO_2, before assaying on a FLIPR-384.

Formate, acetate, propionate and butyrate (Sigma) were prepared in Hank’s balanced salt solution, 25 mmol/L Hepes, and 0.1% bovine serum albumin. The maximum change in fluorescence over baseline was used to determine agonist response. The dose-response curves and EC₅₀ values were obtained by nonlinear regression (Prism 4; Graph Pad Software, San Diego, CA, USA).

PGE₂ was measured by ELISA kit from Assay Designs (Ann Arbor, MI, USA). Cytokines and chemokines were measured by MesoScale ELISA (MesoScale Discovery, Gaithersburg, MD, USA) according to the manufacturer’s instructions. To generate the conditioned media, 1 × 10⁶ cells per mL were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mmol/L L-glutamine, 10 mmol/L Hepes, 50 000 U/L penicillin and 50 μg/mL streptomycin.; and seeded in 24-well plates for stimulation with SCFAs and/or incubation with selective inhibitors (Cayman Chemical, Ann Arbor, MI, USA), including MLnFP (methyl α-linolenyl fluorophosphonate), a phospholipase inhibitor; 1400W, a selective iNOS inhibitor; SC-560, a selective cyclooxygenase-1 (COX-1) inhibitor; CAY10404, a selective COX-2 inhibitor; PTX, pertussis toxin. The tested concentrations of each inhibitor (except PTX) was chosen to be about 100 times of its IC₅₀ as reported in the data sheet provided by the manufacturer.

Sprague-Dawley male rats from Charles River Laboratories, 4 per group and approximately 200 g body weight, were anesthetized with isoflurane gas and the subplantar space of the paw injected with 0.1 mL of 100 mmol/L solutions of the SCFAs, either formate, acetate, propionate or butyrate prepared fresh in 0.9% saline. Lipopolysaccharide (LPS), Escherichia coli 0127:B8 (Sigma) was also injected at 3 μg in saline either alone or in combination with 0.1 mL of 200 mmol/L sodium butyrate. Rats in the normal group were not injected. At 3 h post-injection, the rats were humanely euthanized and a uniform punch biopsy of the injected site was taken from each rat. The punch biopsies were immediately placed in PMSF (phenylmethanesulphonyl fluoride) buffer containing 10 g/L of indomethacin and frozen at -20°C. The tissues were homogenized in this collection buffer and assayed for PGE₂.

All statistical analysis was performed by Mann-Whitney U test using GraphPad Instat version 3.06 for Windows XP (GraphPad Software, San Diego, CA, USA). All studies in animals were performed in accordance with the regulations specified by the National Institutes of Health Principles of Laboratory Animal Care (1985 revised version) and the Schering-Plough Research Institute Animal Care and Use Committee.

Both GPR43 and GPR41 are activated by SCFAs and reported to be expressed in immune cells. To examine the role of GPR43 and GPR41 in human immune cells, we initially quantified their expression levels in human neutrophils and monocytes by Taqman analysis. Human neutrophils and monocytes were each isolated from human donors to 95% purity. Some of them were stimulated with LPS. RNAs were isolated and analyzed for GPR43 and GPR41 expression by Taqman. Figure 1 shows that GPR43 is expressed in both human neutrophils and monocytes at a much higher level than GPR41. It also appears that LPS stimulation did not affect their expression levels.

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Figure 1 GPR43 is highly expressed in human neutrophils and monocytes. Human neutrophils and monocytes were isolated from human whole blood as described in Materials and Methods. Isolated human neutrophils or monocytes were stimulated with 100 ng/mL of lipopolysaccharide (LPS) for 3 h. RNAs were isolated and evaluated by Taqman analysis for absolute quantities of GPR43 and GPR41 mRNA molecules.

To investigate the biological activities of SCFAs, both purified human neutrophils and monocytes were exposed to various concentrations of SCFAs (formate, acetate and propionate) in a calcium flux assay. Formate was used as a negative control for the SCFAs. In addition, IL-8 was included as a positive control for neutrophil activation, while monocyte chemotactic protein-1 (MCP-1) and ATP were used as the positive controls for monocyte activation. Since GPR41 couples to G_i/o only, SCFAs should not cause a calcium flux through this receptor, which was confirmed in a recombinant cell line expressing GPR41 (data not shown). Indeed, the agonist potency profile of the calcium response in human neutrophils (Figure 2A) was consistent with the GPR43 receptor response that has been described[9]. From 8 human donors, acetate had an average EC₅₀ of 58.25 ± 12.44 μmol/L (n = 8), and propionate had an EC₅₀ of 200.6 ± 42.42 μmol/L (n = 8). Formate was inactive and IL-8 had an EC₅₀ about 1-2 nmol/L in human neutrophils. Acetate and propionate achieved the same maximal activation response as IL-8 in human neutrophils using this calcium flux assay.

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Figure 2 Short-chain fatty acids (SCFAs) induce robust calcium flux in human neutrophils, but not in human monocytes. Human neutrophils (A) and monocytes (B) were isolated and exposed to the indicated concentrations of formate, acetate and propionate for a calcium flux assay as described in Materials and Methods. Interleukin-8 (IL-8) is included as a positive control for human neutrophils, and monocyte chemotactic protein-1 (MCP-1) and ATP for human monocytes. The dose-response curves and EC₅₀ values were obtained by nonlinear regression using Graph Pad Prism 4 software.

On the other hand, Figure 2B shows that acetate and propionate, up to 100 mmol/L, did not induce significant calcium flux in human monocytes, while MCP-1 and ATP were able to induce robust calcium flux in the human monocyte preparation. It appears that the response of GPR43 to SCFAs for calcium flux is dependent either on cell type or on the state of differentiation of the cells. Indeed, it has been reported that propionic acid induced calcium mobilization in human neutrophils, but not in monocytes, platelets and lymphocytes[12].

To further explore the biological effects of SCFAs, human monocytes were isolated and incubated overnight with various concentrations of SCFAs in the presence or absence of LPS (100 ng/mL) as a proinflammatory stimulus. The supernatants were assayed for lipid mediators such as PGE₂, proinflammatory cytokines and chemokines by either ELISA or Meso Scale Multi-Spot Discovery Technology. Dexamethasone was included in the experiment as a positive control for the induction of inhibition. Figure 3A shows that SCFAs alone strongly enhanced PGE₂ production in human monocytes across a panel of 10 human donors in a concentration-dependent manner. Furthermore, SCFAs synergistically enhanced the PGE₂ production induced by LPS in human monocytes (10 human donors), as shown in Figure 3B. The rank order of potency was butyrate > propionate > acetate, while formate was inactive. Dexamethasone at 100 nmol/L potently inhibited basal PGE₂ and LPS-induced PGE₂ production (Figure 3A and B). Similar results were obtained from 10 human donors. The culture supernatants were also assayed for PGI₂, leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂). SCFAs had no significant effect on these lipid mediators (data not shown), suggesting its effect on PGE₂ production is specific.

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Figure 3 SCFAs strongly induce PGE₂ production in human monocytes and the effect is receptor-mediated. Freshly isolated human monocytes (1 × 10⁹ cells/L) were incubated with the indicated concentrations of SCFAs or 100 nmol/L dexamethasone (Dex), and then cultured for 22 h without (A) or with (B) 100 ng/mL LPS. In a separate experiment (C, D), human monocytes were incubated with the indicated concentrations (in the parentheses) of inhibitors (described below) for 30 min, then with or without 2 mmol/L butyrate for another 30 min, and finally cultured for 22 h without (C) or with (D) 100 ng/mL LPS. The media and DMSO samples were used as controls with no inhibitors. All culture supernatants were assayed for PGE₂ by ELISA. MLnFP (methyl α-linolenyl fluorophosphonate): Phospholipase inhibitor; 1400W: Selective iNOS inhibitor; SC-560: Selective cyclooxygenase-1 (COX-1) inhibitor; CAY10404: Selective COX-2 inhibitor; PTX: Pertussis toxin. (Cayman Chemical, Ann Arbor, MI). The tested concentrations of each inhibitor (except PTX) were chosen to be about 100 times its IC₅₀ as reported in the data sheet provided by the manufacturer.

To examine possible mechanisms of PGE₂ production induced by SCFAs, human monocytes were preincubated with several inhibitors and then added to culture with butyrate and/or LPS. Figure 3C and D show that the PGE₂ production induced by butyrate is inhibited by pretreatment with PTX, COX-1 and phospholipase inhibitors either in the absence or presence of LPS. In contrast, COX-2 and iNOS inhibitors had no effect on PGE₂ production induced by butyrate. The sensitivity to PTX suggests PGE₂ production induced by butyrate is receptor-mediated.

The same culture supernatants described above were also analyzed for 8 proinflammatory cytokines including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α (Meso Scale Multi-Spot Discovery Technology). In the absence of LPS, SCFAs had no effect on the level of any of the 8 cytokines including IL-10, as shown in Figure 4A. In contrast, SCFAs specifically inhibited IL-10 production stimulated with LPS (Figure 4B), but had no effect on the other 7 cytokines with LPS stimulation (including LPS-induced production of IL-1β and TNF-α. The effect of dexamethasone on the cytokine profile is consistent with what has been previously described; briefly, it inhibited LPS-induced production of IL-1β, IL-10 and TNF-α.

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Figure 4 SCFAs specifically inhibit LPS-induced IL-10 production and constitutive MCP-1 expression in human monocytes. The culture supernatants as in Figures 3A and B were analyzed for 8 proinflammatory cytokines (including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α) or 9 proinflammatory chemokines (including MCP-1, MCP-4, eotaxin, eotaxin-3, IL-8, IFN-γ inducible protein-10, macrophage inflammatory protein-1β, macrophage-derived chemokine, thymus and activation-regulated chemokine) by Meso Scale Multi-Spot Discovery Technology. Only the data for IL-10 (A, B) and MCP-1 (C, D) are shown here. Similar results were obtained from 10 human donors.

It is consistently observed that isolated human monocytes produce high levels of MCP-1 in the culture supernatants without stimulation. Figure 4C and D show that SCFAs inhibited this constitutive MCP-1 production in a concentration-dependent manner either in the absence or presence of LPS. SCFAs did not inhibit the constitutive high levels of eotaxin-3, macrophage-derived chemokine and macrophage inflammatory protein-3β in the cultures, suggesting that the effect of SCFAs on MCP-1 production is specific. In this experiment, 20 mmol/L formate showed some inhibition, which is likely the result of a nonspecific effect.

To further confirm the effect of SCFAs, human PBMC were isolated by Ficoll-Hypaque centrifugation procedure and incubated with SCFAs and/or LPS. The culture supernatants were analyzed as before for PGE₂, cytokines and chemokines. The effects of SCFAs on PGE₂, MCP-1 and IL-10 production were similar to that observed in the isolated human monocytes. However, as shown in Figure 5, SCFAs inhibited LPS-induced production of TNF-α and IFN-γ in human PBMC in a concentration-dependent manner, an effect not observed in isolated human monocytes where IFN-γ production was low and not stimulated by LPS addition. In the human PBMC experiment, dexamethasone inhibited LPS-induced production of IL-1β, IL-10, IFN-γ and TNF-α.

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Figure 5 SCFAs inhibit LPS-induced production of TNF-α and IFN-γ in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Human PBMC isolated by Ficoll-Hypaque centrifugation procedure (1 × 10⁶ cells/mL) were incubated with the indicated concentrations of short-chain fatty acids (SCFA) or 100 nmol/L dexamethasone (Dex), and then cultured for 22 h with or without 100 ng/mL LPS. Culture supernatants were analyzed for 8 proinflammatory cytokines including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α by Meso Scale Multi-Spot Discovery Technology. The data for TNF-α (A) and IFN-γ (B) with LPS stimulation are shown here.

To demonstrate the effect of SCFAs in vivo, SCFAs and LPS alone or in combination were injected by the intraplantar route into a rat paw. This was done to initiate an inflammatory response and determine what effect the butyrate would have on lipid mediator and cytokine induction. In the representative experiment shown in Figure 6, acetate, propionate and butyrate alone could induce PGE₂ production in rat paw. Formate was inactive and the level of PGE₂ was equivalent to that extracted from a normal (not injected) paw. In addition, suboptimal LPS also induced PGE₂ production and the combination of LPS and butyrate strongly enhanced PGE₂ production in a synergistic manner (P < 0.01). These in vivo data, although preliminary in nature, support the effects of SCFAs on PGE₂ production observed in human monocytes and PBMC. Cytokine analysis was performed on the tissue homogenates but no clear pattern emerged that was similar to that obtained in the human monocytes and PBMC in vitro experiments. Sodium butyrate is extremely labile with a very high diffusion rate. Maintenance of a sufficient concentration in the paw subcutaneous space was a significant problem in these experiments.

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Figure 6 SCFAs induce PGE₂ production by intraplantar injection into rat paws. The paw of male Sprague-Dawley rats was injected intraplantarly with 0.1 mL of 100 mmol/L formate, acetate, propionate or butyrate. Three micrograms of LPS in saline was also injected alone or in combination with 0.1 mL of 200 mmol/L butyrate. There were 4 rats per group. The normal group was not injected. Three hours post injection, rat paws were punched and fluids were collected in phenylmethanesulphonyl fluoride buffer with indomethacin. All punch fluids were assayed for PGE₂ by ELISA. P < 0.01 for butyrate + LPS group vs normal group, Mann-Whitney U test.

Short-chain fatty acids (SCFAs) are produced by bacterial fermentation of dietary fiber in the hindgut in millimolar concentrations. They have long been known to modulate the immune response and have a beneficial effect in the colon and on colitis.

The molecular mechanism for the effect of SCFAs in immune cells of the gastrointestinal tract has not been characterized. Recently, a free fatty acid receptor GPR43 was found to be highly expressed in both neutrophils and monocytes and identified to be activated by SCFAs. In neutrophils, SCFAs induce calcium flux and chemotaxis. However, the effect of SCFAs in monocytes is of high interest, but is largely unknown.

This study showed that SCFAs can induce specific and robust prostaglandin E₂ (PGE₂) production either alone or in synergy with lipopolysaccharide in human monocytes and peripheral blood mononuclear cells (PBMC). Furthermore, SCFAs can specifically regulate production of monocyte chemotactic protein-1, interleukin-10, tumor necrosis factor-α and interferon-γ in human monocytes and/or PBMC. Finally, we showed that SCFAs can induce PGE₂ production in vivo by intraplantar injection into rat paws.

The present findings of antiinflammatory activities of SCFAs in immune cells expand the database on these fatty acids and may help explain the known beneficial effects of SCFAs in the colon and on colitis. Future studies are needed in knockout mice and with specific small molecules to prove the exact molecular target for these effects of SCFAs. The authors speculate that the action of SCFAs is most likely through activation of the free fatty acid receptor GPR43, a potential therapeutic target for the development of an innovative treatment of colitis.

This is a study in an important area of inflammatory activities of SCFAs and its effects on the related cytokines and chemokines. In the present paper, the authors examined the inflammatory activities of SCFAs and the production of the related receptors and analyzed the mechanisms. The experiments were well organized.